Purine nucleoside 5&#39;-phosphate (mono, di or tri) 3&#39;(2&#39;)-diphosphates and processes for their preparation

ABSTRACT

New purine nucleoside 5&#39;-phosphate (mono, di or tri) 3&#39;(2&#39;)-diphosphates and the sodium, lithium and potassium salts thereof are elaborated by the enzymatic transfer of pyrophosphoryl group from ATP (adenosine triphosphate), dATP (deoxyadenosine triphosphate) and pppApp (adenosine 5&#39;-triphosphate 3&#39;(2&#39;) diphosphate) to specified 5&#39;-purine nucleotides, and a new nucloetide pyrophosphotransferase used for this enzyme reaction is produced by actinomycetes and other microorganisms and recovered from the culture filtrate and mycelium by the conventional methods for recovering enzymes. These compounds are useful for treating leukemia L1210 in mice.

This is a division of application Ser. No. 559,462 filed Mar. 18, 1975now U.S. Pat. No. 4,059,487.

BACKGROUND OF THE INVENTION

This invention relates to new purine nucleoside 5'-phosphate (mono, dior tri) 3' (2')-diphosphates and the lithium, sodium and potassium saltsthereof, and to methods for the production of the purine nucleoside5'-phosphate (mono, di or tri) 3' (2')-diphosphates. More particularly,it relates to new purine nucleoside 5'-phosphate (mono, di or tri) 3'(2')-diphosphates which are produced by the enzymatic transfer ofpyrophosphoryl group from ATP, dATP and pppApp to specified 5'-purinenucleotides using microorganisms belonging to the genera Streptomyces,Actinomyces and Streptoverticillium, or a new nucleotidepyrophosphotransferase thereof, and to processes for the production ofthe purine nucleoside 5'-phosphate (mono, di or tri) 3'(2')-diphosphates. It also relates to a new nucleotidepyrophosphotransferase, and to the methods for its recovery andpurification from the cultured broth and mycelium of microorganismsbelonging to the genera Streptomyces, Streptoverticillium andActinomyces.

As is well known, adenosine tetraphosphate (ppppA), guanosinetetraphosphate (ppGpp) and Guanosine pentaphosphate (pppGpp) aredistributed in nature, especially in microorganisms and animals, asunusual nucleoside phosphates. The ppGpp and pppGpp are particularlybeing watched with keen interest in the regulation of protein andribonucleic acid syntheses in E. coli, and it is expected that they havean important role in glycolysis, lipid synthesis, energy metabolism andphosphorylation of various organisms.

In the present invention, purine nucleoside 5'-phosphate (mono, di ortri) 3' (2')-diphosphate compounds are generalized as the followingsymbol:

    mXpp

in which X represents one of the nucleosides selected from adenosine(abbreviated as A), guanosine (abbreviated as G) and inosine(abbreviated as I), and p and p (CH₂)p stands for phosphate residue andmethylene phosphate residue, respectively. In this specificationphosphate residue is also referred to as phosphoryl group.

The notation, m represents a number of phosphoryl groups at the5'-position of the nucleoside in which m is an integral number from 1 to3. The p after the X is the 3' (2')-position. The notation 3' (2')-indicates that the position of the phosphoryl group attached to theribose is either 3'- or 2'- of the ribose, as there is the reversibleexchange of the phosphoryl group between the 3'- and the 2'- position ofribose in the nucleoside under a certain condition. For instance, pppAppindicates adenosine 5'-triphosphate 3' (2')-diphosphate and p(CH₂)ppGppis β, γ-methylene guanosine 5'-triphosphate 3' (2')-diphosphate.

However, it is difficult to extract unusual purine nucleoside phosphatessuch as ppGpp and pppGpp from microorganisms because of a very smalldistribution therein, and further the chemical synthesis of thesenucleoside phosphates is not established yet. Biochemical syntheses ofppGpp and pppGpp are possible using E. coli ribosome, ATP and GDP orGTP, but this method is not suitable for industrial production of largeamounts, because of the complicated processes and extremely low yield.Other new nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphatessuch as pppApp, ppApp, pppIpp, etc. cannot be produced by E. coli. Inorder to investigate profoundly the physiological role of thesenucleotides in organisms, development of an economical and simple methodfor their preparation has been awaited. ATP is the most importantsubstance in phosphorylation and energy metabolism of organisms, andrecently it was shown that E. coli and B. subtilis can synthesisenzymatically the characteristic nucleotides, ppGpp and pppGpp, whichparticipate in nucleic acid and protein syntheses, from ATP and GDP orGTP. However, the purification of the enzyme playing a part in theformation of ppGpp and pppGpp has been unsuccessful so far. A number ofphosphotransferases and nucleotide-degrading enzymes have been isolatedfrom microorganisms and characterized, but the enzyme concerned with theformation of the aforementioned nucleoside phosphates has not beenreported yet.

SUMMARY OF THE INVENTION

We have aimed toward the use of purine nucleoside phosphate compounds asbiochemical reagents, high energy phosphate additives and medicines, andhave continued to search through a wide variety of natural sources formicroorganisms possessing a potential nucleotide pyrophosphotransferaseactivity. As a result of screening, a new species belonging toStreptomyces, Streptomyces adephospholyticus nov. sp. A4668, whichpossesses a potential nucleotide pyrophosphotransferase activity wasisolated, and the enzyme which produces pppGpp, ppApp, pppIpp, etc. bycatalyzing the transfer of pyrophosphoryl group from ATP, dATP andpppApp to ATP, GTP, GDP, IDP, AMP, etc. was purified to a substantiallyhomogeneous entity from the culture filtrate. Furthermore, the presentinventors discovered the occurrence of nucleotide pyrophosphotransferasein several known species of actinomycetes, and succeeded in readilyproducing the nucleoside 5'-phosphate (mono, di or tri) 3'(2')-diphosphates by means of the characteristic transfer ofpyrophosphoryl group at the 5'-position of ATP, dATP and pppApp topurine nucleoside phosphates such as AMP, ADP, ATP, GMP, GDP, IDP, ITP,etc. using the cultured broth and mycelium of nucleotidepyrophosphotransferase -- producing microorganisms. In addition, thepresent invention provides a method for the preparation andcharacterization of the enzyme.

Herein

Amp is adenosine 5'-monophosphate

Adp is adenosine 5'-diphosphate

Gmp is guanosine 5'-monophosphate

Imp is inosine 5'-monophosphate

Idp is inosine 5'-diphosphate

Itp is inosine 5'-triphosphate

p(CH₂)ppA is β,γ-methylene adenosine 5'-triphosphate

p(CH₂)ppG is β,γ-methylene guanosine 5'-triphosphate

ppApp is adenosine 5'-diphosphate 3' (2')-diphosphate

Furthermore, from the results of extensive studies on the physiologicalrole of nucleotides in organisms and the distribution and biochemistryof the enzyme, the present inventors found that the nucleotidepyrophosphotransferase stimulates or inhibits the growth of varioustumor cells in culture and also exhibits potent antitumor activity.Furthermore, the new nucleotide 5'-phosphate (mono, di or tri) 3'(2')-diphosphates inhibit the growth of leukemic L1210 cells in mice andprevent thrombus formation.

The present invention was achieved on the basis of discoveries ofmedical uses such as chemotherapeutic and diagnostic agent of thesenucleotides.

It is an object of the present invention to provide new nucleoside5'-phosphate (mono, di or tri) 3' (2')-diphosphates and their saltsexpressed as the following general formula:

    mXpp

It is another object to provide a method for producing new nucleoside5'-phosphate (mono, di or tri) 3' (2')-diphosphates as described aboveby the enzymatic transfer of a pyrophosphoryl group from specifiednucleotides to other specified nucleotides using microorganisms or anenzyme therefrom. It is a further object to provide new nucleoside5'-phosphate (mono, di or tri) 3' (2')-monophosphates lacking onephosphate at the 3' (2')-position of the above described nucleoside5'-phosphate (mono, di or tri) 3' (2')-diphosphates. It is a stillfurther object to provide a new nucleotide pyrophosphotransferasecharacteristically catalyzing the pyrophosphoryl transfer reaction.

Still another object is the provision of a process for making thecharacteristic nucleotide pyrophosphotransferase using microorganisms.Other objects are to provide a chemotherapeutic agent for cancer and onefor the use in inhibiting or preventing thrombus formation in, forexample, postoperative situations and conditions where platelet adhesionis excessive. This will be evident from the following detaileddiscussion of our invention.

BRIEF EXPLANATION OF THE DRAWINGS

FIGS. 1 to 3 respectively show ultraviolet absorption spectra ofadenosine 5'-triphosphate 3'-diphosphate, guanosine 5'-diphosphate3'-diphosphate and inosine 5'-triphosphate 3'-diphosphate in H₂ O, inwhich the vertical axis represents optical density.

FIGS. 4 and 5 are NMR spectra of guanosine 5'-diphosphate 3'-diphosphateand inosine 5'-triphosphate 3'-diphosphate respectively.

FIG. 6 is ¹³ C NMR spectrum of adenosine 5'-triphosphate 3'-diphosphatein D₂ O (100 MHz).

FIG. 7 shows the time course of enzyme production by (A) Streptomycesmorookaensis ATCC 19166 and (B) Streptomyces aspergilloides ATCC 14808,in which the vertical axis represents ml. of P.C.V. (packed cell volume)and pH (right) and enzyme activity (left) and the horizontal axisrepresents hours.

FIG. 8 shows the relationship of enzyme concentration and thermalstability of the enzyme obtained from Streptomyces morookaensis ATC19166, in which vertical axis represents % of residual activity.

FIG. 9 shows the molecular weight of the enzymes obtained from themicroorganisms according to this invention, calculated by using SephadexG-75 gel filtration and standard proteins as controls in which thevertical axis represents molecular weight (X 10⁴) and the horizontalaxis represents the fraction number.

DETAILED EXPLANATION OF THE INVENTION Microorganisms and the methods fortheir cultivation Microorganisms:

The microorganisms used in the present invention are a new species ofStreptomyces, Streptomyces adephospholyticus, nov. sp. A4668 and knownspecies of actinomycetes belonging to the genera Streptomyces,Actinomyces and Streptoverticillium. Since the actinomycetes are easilymutable naturally or artifically, the microorganisms in the presentinvention include the typical strain and all the natural and artificialvariants and mutants thereof. That is, the microorganisms of the presentinvention include all strains of actinomycetes possessing nucleotidepyrophosphotransferase activity.

The following strains are cited as examples:

Streptomyces aspergilloides ATCC 14804

streptomyces morookaensis ATCC 19166

streptomyces hachijoensis ATCC 19769

streptoverticillium septatum ATCC 27464

actinomyces violascens ATCC 23968

The cultures of these strains have been deposited in the American TypeCulture Collection, Rockville, Md., and anyone can obtain the strainsunder the prescribed procedures.

One of the strains used in the present invention, Streptomycesadephospholyticus nov. sp. A4668 was newly isolated from a soil samplecollected at Sakai-city, Osaka, Japan, and shows the followingmorphological, macroscopic, microscopic and biochemical properties:

(a) Morphological characteristics (Incubated on a malt yeast extractagar medium at 28° C. for 10 to 20 days).

Aerial mycelia were simply branched, and at the tips of hyphaesporophores formed long open spirals. Mature spore chain was long andbore more than 10 spores per chain.

Spores were oval with spiny surface, and measured 0.4-0.6 × 0.6-0.8μ andthe presence of flagella, sporangia or sclerotia was not observed.

Incubation was carried out at 28° C. unless otherwise specified.

(b) Cultural characteristics on various media:

(1) On sucrose -- nitrate agar:

Growth: slow or moderate

Aerial mycelium: Partly velvety, white with a purplish tinge

Substrate mycelium: White to pale yellow, partly pale yellow-orange

Soluble pigment: none

(2) On glucose-asparagine agar:

Growth: moderate and wrinkled

Aerial mycelium: partly velvety, white with a purplish tinge

Substrate mycelium: light yellow

Soluble pigment: none

(3) On glycerin -- asparagine agar:

Growth: moderate

Aerial mycelium: partly velvety, at first a white with pink, after twoweeks of cultivation a light grayish-purple

Substrate mycelium: at first a light yellow orange, later a light grayreddish-brown

Soluble pigment: usually none, sometimes a pale yellow after two weeksof cultivation

(4) On starch agar (starch-inorganic salts agar):

Growth: moderate

Aerial mycelium: partly pointed, white to grayish purple or purple orpurplish white on old culture.

Substrate mycelium: white to pale yellow in young culture, lightyellow-orange in old culture.

Soluble pigment: none

(5) On tyrosine agar:

Growth: moderate

Aerial mycelium: abundant, white, very light purple in old culture

Substrate mycelium: grayish yellow to rose beige or moderate yellowishpink, light brown in old culture

Soluble pigment: yellow to grayish yellow-brown, disappeared in oldculture

(6) On nutrient agar:

Growth, moderate, surface wrinkled

Aerial mycelium: none

Substrate mycelium: light gray reddish brown

Soluble pigment: light brown which disappeared in old culture

(7) On yeast extract-malt extract agar:

Growth: abundant, wrinkled

Aerial mycelium: velvety, white to pale yellow

Substrate mycelium: pale yellow to grayish yellow, moderate yellowishpink to light gray reddish brown in old culture

Soluble pigment: none

(8) On oat meal agar:

Growth: abundant, wrinkled

Aerial mycelium: white, changing to grayish yellowish pink or purplishpink in old culture

Substrate mycelium: grayish yellow in young culture, light grayishyellow-brown to light brown in old culture

Soluble pigment: none

(c) Physiological characteristics:

(1) Growth temperature:

Optimal temperature is 25° C. to 30° C., and scant growth at 10° C., butno growth above 37° C.

(2) gelatin liquefaction on glucose-peptone-gelatin medium at 20° C.:positive

(3) Starch hydrolysis on starch-inorganic salts agar: positive

(4) Peptonization and coagulation of skimmed milk: positive

(5) Melanin formation on tyrosine agar, peptone yeast extract Fe⁺⁺ agar,and tryptone yeast extract broth: positive

(6) Nitrate reduction: positive

(7) Utilization of carbohydrates on Pridham-Gottlieb basal medium:

Abundant growth with L-arabinose, D-xylose, D-glucose, D-fructose,inositol, raffinose and D-mannitol; slight growth with sucrose; nogrowth or very slight growth with L-rhamnose

Classifying on the basis of the morphological and physiologicalcharacteristics of the strain according to "The Actinomycetes", Vol. II,1961, by S. A. Waksman, Bergey's Manual of Determinative Bacteriology,7th edition, 1957, and International Journal of Systematic Bacteriology,Vol. 18 (1968) No. 2 and 4, and Vol. 19 (1969) No. 4, the strain belongsto the genus Streptomyces, because vegetative mycelia were notsegregated into bacillary fragments in the liquid media and aerialmycelia were simply branched with the sporophores forming long openspirals at the tips of hyphae.

Comparing these microbiological characteristics with those of knownspecies of Streptomyces, the present strain apparently resembles theLavendulae series, Streptomyces lavendulae and St. venezualae but isdifferent from those strains in detail.

Among known species, the cultural and physiological characteristics ofthe present strains were very similar to Actinomyces violascens. Fromthe results of parallel cultures of the present strain and the standardstrain of Act. violascens ISP 5183, some differences were found in thecultural characteristics such as the color of aerial and substratemycelia and appearances on yeast extract malt extract agar, tyrosineagar, peptone-yeast extract-Fe⁺⁺ agar, nutrient agar media, and also inthe carbon assimilation patterns. The present strain could assimilateD-mannitol, inositol and fructose, but Act. violascens could notassimilate D-mannitol and only weakly was able to utilize the other twosugars.

From the results mentioned above, the present strain was identified as anew species and named St. adephospholyticus nov. sp. A 4668.

A culture of St. adephospholyticus A 4668 was deposited in theFermentation Research Institute, Japan, on 24, March, 1973, and in theAmerican Type Culture Collection, Rockville, Md., and added to itspermanent collection of microorganisms at ATCC No. 31122 and FERM No.1992, respectively.

Cultivation of the microorganisms possessing nucleotidepyrophosphotransferase activity:

The new species, St. adephospholyticus, and other authentic strains ofactinomycetes possessing nucleotide pyrophosphotransferase activityinclude the enzyme constitutively, and are characterized by the factthat they excrete the enzyme in the culture filtrate extracellurallyunder the general fermentation conditions used for the cultivation ofother actinomycetes. Although cultivation on a solid medium and recoveryfrom mycelium are possible in the preparation of the enzyme, recoveringthe enzyme which has been actively excreted in the culture filtrate bythe submerged aerobic culture is especially advantageous industrially inthe production of large quantities of the pure enzyme preparation.

Media consisting of known kinds of nutritional sources for the growth ofactinomycetes can be used for the production of nucleotidepyrophosphotransferase. As the source of carbon, the medium preferablycontains glucose, sucrose, maltose, fructose, starch, starchhydrolysate, glycerol, glycine, alanine, glutamic acid, molasses,dextrin, oil, fats and the like; as the source of nitrogen, the mediumpreferably contains organic materials such as peptone, meat extract,yeast extract, soybean meal, fish meal, casein hydrolysate and urea, andinorganic sources of nitrogen such as nitrates and ammonium salts, e.g.ammonium sulfate and ammonium chloride; and the medium also containsother inorganic salts such as sodium chloride, potassium chloride,potassium phopshate, magnesium sulfate, and calcium carbonate and traceamounts of heavy metals such as mangenese, iron, zinc and the like. Inaerated submerged cultures, an antifoam such as liquid paraffin, soybeanoil, fatty oils or silicone is used.

The culturing temperature is usually 20° C. to 40° C., with thepreferred range of temperature being 25° to 30° C. It is not necessaryto control the pH of the culture medium during the cultivation; theenzyme activity in the culture filtrate reaches a maxiumum at 42 to 72hours of incubation.

Enzyme and method of purification thereof:

Purification:

An example for the production of the enzyme is described below.

A medium (100 ml) consisting of glycerol 2%, polypeptone 4%, KH₂ PO₄0.1%, K₂ HPO₄ 0.1%, MgSO₄.7H₂ O 0.04%, Mn⁺⁺ and Fe⁺⁺ 2ppm each wasplaced in 500 ml Sakaguchi -- shaking flasks and sterilized at 120° C.for 15 minutes. To this sterilized medium, Streptomyces aspergilloidesATCC 14808 and St. morookaensis ATCC 19166 were inoculated into separateflasks from an agar slant culture by means of a platinum loop, andincubated on a reciprocal shaker at 28° C. The time course of the enzymeproduction in the culture filtrate, as shown in FIG. 7, was determinedby the method described later.

FIG. 7A shows the time course of enzyme activity of St. morookaensisATCC 19166 and FIG. 7B is that of St. aspergilloides ATCC 14808, inwhich enzyme activity is expressed as units/ml. determined by theformation of pppApp or Pi (inorganic phosphate), and cell growth isexpressed as P.C.V. (packed cell volume).

After fermentation, the enzyme can be purified and recovered byconventional methods such as salting-out with ammonium sulfate, solventprecipitation, extraction, adsorption, electrophoresis, electrofocusing,gel-filtration, the use of ion-exchange resins and the like or by acombination of methods where at least one or more are selected fromthese.

Under cultivation conditions capable of growing microorganisms, theenzyme is found mainly in the liquid part of the culture broth after themycelia have been removed. Obtaining the enzyme in pure form from theculture filtrate where it has accumulated in high concentration isadvantageous from an industrial viewpoint.

Purification and preparation processes of the enzyme from St.morookaensis ATCC 19166 is cited as an example in Table 1.

Purity of the final enzyme preparation was examined by polyacrylamideand SDS-polyacrylamide gel electrophoreses, and the purified enzymepreparation migrated as a single band on the gels showing the enzyme tobe electrophoretically homogeneous.

                                      Table 1                                     __________________________________________________________________________    Purification processes of nucleotide pyrophosphotransferase                   from Streptomyces morookaensis ATCC 19166                                                      Total                                                                              Total                                                               Volume                                                                             activity                                                                           protein                                                                              Specific                                                                            Yield                                      Processes   (ml) (units)                                                                            (OD.sub.280nm)                                                                       activity                                                                            (%)                                        __________________________________________________________________________    Culture filtrate                                                                          20000                                                                              77190                                                                              1218420                                                                              0.63  100                                        (NH.sub.4).sub.2 SO.sub.4 ppt.                                                60% saturation                                                                            9789 471811                                                                             256220 1.84  61.1                                       DEAE-cellulose                                                                column (pH 5.6)                                                                           11200                                                                              436800                                                                             146940 2.98  56.6                                       CM-Sephadex C-50                                                              column (pH 5.6)                                                                           1660 72422                                                                              16384  4.42  9.4                                        CM-Sephadex C-25                                                              column (pH 5.5)                                                                           910  16244                                                                              309    52.6  2.1                                        DEAE-Sephadex A-25                                                            (pH 8.7)    159  6298 20     315   0.8                                        Sephadex G-75                                                                 (pH 8.7)    14   6019 6.7    903   0.8                                        __________________________________________________________________________

The quite low yield of purified enzyme in the example was caused by aconsiderable loss of enzymatic activity with the removal of impureprotein during the purification process and with the rapid inactivationby dilution.

The addition of proteins such as casein and bovine serum albumin andnon-ionic surface active agents such as Tween and Span to the enzymesolution protected the enzyme against inactivation, improved the yieldof the purified enzyme and made it possible to store the enzyme for along time.

Enzyme assays were carried out using either of the following methods:

(1) Inorganic phosphate determination by a modification of Nakamura'smethod: The standard reaction mixture contained 0.25 M glycine-NaOHbuffer (pH 10.0), 0.2 ml.; 0.05 M MgCl₂, 0.05 ml.; 0.05 M ATP, 0.05 ml.;the enzyme solution (to be diluted by buffer if necessary), 0.1 ml.; anddistilled water, 0.1 ml. in a final volume of 0.5 ml. After incubationat 37° C. for 10 minutes, the reaction was terminated by adding 0.5 ml.of 0.2 N acetic acid, and chilling on ice. Immediately, 3 ml. ofdeveloping reagent (amidol-molybdate) was added and the mixture wasallowed to stand for 10 minutes at 37° C. The optical density wasmeasured at 750 nm and the moles of Pi released were calculated usingthe standard curve for Pi. A unit of enzyme activity is defined as theamount of enzyme which is capable of forming 1μ mole of Pi per minuteunder standard conditions.

(2) Determination of pppApp and AMP formed in the reaction mixture usingradioactive ATP.

The reaction mixture contained 20 mM adenosine-8-3H-triphosphate (2mCi/m mole), 5 mM MgCl₂, 62.5 mM glycine-NaOH buffer (pH 10.0) and 0.1ml. of enzyme solution to give a total volume of 0.2 ml. The mixture wasincubated at 37° C. for 10 minutes and the incubation was terminated byaddition of 0.01 ml. of 1 N acetic acid. Twenty microliters of thereaction mixture were spotted on a polyethyleneimine (PEI) cellulose Fthin layer sheet (E. Merck AG), dried and developed in 0.75 M KH₂ PO₄,pH 3.4, for two hours at room temperature. After chromatography thesheets were dried and exposed to ultraviolet light provided by a ManasulUV lamp to localize the pppApp and AMP formed. The spots on thechromatograms corresponding to pppApp and AMP were cut out and placed ina scintillation vial containing 0.5 ml. of 0.1 N HCl. Vials were boiledfor 15 minutes to elute nucleotides adsorbed on the PEI-cellulose andradioactivity was measured by 10 ml. of BRAY Solution in a liquidscintillation counter. A unit of enzyme activity is defined as theamount of enzyme forming 1μ mole of pppApp or AMP per minute under theabove condition.

Enzyme:

The nucleotide pyrophosphotransferase preparation obtained from themicroorganisms described in the present invention by a combination ofthe various above-mentioned methods was demonstrated to be a new enzymepossessing the following physicochemical properties;

(a) Enzyme reaction and substrate specificity:

The enzymes obtained from the above-mentioned six microorganismscatalyze the transfer reaction of a pyrophosphoryl group from ATP, dATPand pppApp to 3' (2')-position of AMP, ADP, ATP, GMP, GDP, GTP, IMP,IDP, ITP, p(CH₂)ppA, p(CH₂)ppG and the like. The new reaction productsare pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp, pIpp, ppIpp, pppIpp,p(CH₂)ppApp, p(CH₂)ppGpp and the like.

Other pyrimidine nucleotides, paranitrophyenylphosphate, pyrophosphate,glucose-1-phosphate, glycerin-3-phosphate, tripolyphosphate,nucleosides, and bases cannot accept or donate phosphoryl group. Thereare some differences in pyrophosphate acceptor and donor activitiesdepending upon pH, metal ions and microorganisms, but generally thepreference of phosphate donor activity is in the order of ATP, pppAppand dATP, and that of phosphate acceptor activity is in the order ofadenosine phosphate, guanosine phosphate and inosine phosphate.

(b) pH optimum and metal requirements:

The enzyme capable of catalyzing the formation of pppApp from ATPexhibited an absolute requirement for divalent metal ions, and noactivity in the reaction mixture is detected in the absence of divalentmetal ions. The pH optimum and optimal concentrations of divalent ionsdepend upon specific metal ions and enzyme sources as shown in Table 2.

                                      Table 2                                     __________________________________________________________________________    Relationship between pH and metal ions on the enzyme activity                                  Optimum         Relative                                                      concentration                                                                           Optimum                                                                             activity                                     Enzyme sources                                                                           Metal ions                                                                          (M)       pH    (%)                                          __________________________________________________________________________               Mg.sup.++                                                                           5 × 10.sup.-3                                                                     10 - 10.5                                                                           100                                          Actinomyces                                                                              Mn.sup.++                                                                           3 × 10.sup.-3                                                                     9.5 - 10                                                                            45                                           violascens Co.sup.++                                                                           5 × 10.sup.-3                                                                     9     43                                           ATCC 23968 Zn.sup.++                                                                           5 × 10.sup.-3                                                                     9.5   15                                                      Fe.sup.++                                                                           5 × 10.sup.-3                                                                     9     30                                                      Mg.sup.++                                                                           10.sup.-2 5 × 10.sup.-3                                                           9.5 - 10                                                                            100                                          Streptoverticillum                                                                       Mn.sup.++                                                                           10.sup.-2 10 - 10.5                                                                           40                                           septatum   Co.sup.++                                                                           7 × 10.sup.-3                                                                     8 - 8.5                                                                             35                                           ATCC 27464 Zn.sup.++                                                                           5 × 10.sup.-3                                                                     8.5 - 9                                                                             20                                                      Fe.sup.++                                                                           5 × 10.sup.-3                                                                     7.5 - 8                                                                             20                                                      Mg.sup.++                                                                           10.sup.-2 5 × 10.sup.-3                                                           10 - 11                                                                             100                                          Streptomyces                                                                             Mn.sup.++                                                                           3 × 10.sup.-3                                                                     11    60                                           morookaensis                                                                             Co.sup.++                                                                           7 × 10.sup.-3                                                                     9 - 9.5                                                                             65                                           ATCC 19166 Zn.sup.++                                                                           5 × 10.sup.-3                                                                     9.5   15                                                      Fe.sup.++                                                                           5 × 10.sup.-3                                                                     8 - 9 25                                                      Mg.sup.++                                                                           5 × 10.sup.-3                                                                     10 - 11                                                                             100                                          Streptomyces                                                                             Mn.sup.++                                                                           7 × 10.sup.-3                                                                     10.5  50                                           aspergilloides                                                                           Co.sup.++                                                                           7 × 10.sup.-3                                                                     8.5 - 9                                                                             60                                           ATCC 14808 Zn.sup.++                                                                           5 × 10.sup.-3                                                                     9 - 10                                                                              20                                                      Fe.sup.++                                                                           5 × 10.sup.-3                                                                     8.5   55                                                      Mg.sup.++                                                                           10.sup.-2 5 × 10.sup.-3                                                           9.5   100                                          Streptomyces                                                                             Mn.sup.++                                                                           5 × 10.sup.-3                                                                     9 - 9.5                                                                             35                                           hachjioensis                                                                             Co.sup.++                                                                           7 × 10.sup.- 3                                                                    8 - 8.5                                                                             75                                           ATCC 19769 Zn.sup.++                                                                           2 × 10.sup.-3                                                                     8.5 - 9                                                                             20                                                      Fe.sup.++                                                                           5 × 10.sup.-3                                                                     7.5   25                                           __________________________________________________________________________

(c) Optimal temperature:

There is no difference in the optimal temperature of the enzyme reactionirrespective of the kind of microorganisms employed. The temperature of40° to 45° C. is optimal under optimal pH in the presence of magnesiumion.

(d) Stability

Enzyme isolated from six strains of actinomycetes are similarly stableat a pH ranging from 7 to 11 and unstable at an acidic pH below 6. Whenthe enzyme solution has stood at 37° C. for two hours, residual activityis 10 to 20% at pH 4, 30 to 50% at pH 5, and 60 to 70% at pH 6. Althoughthermal stability is influenced by the degree of dilution of the enzymesolution and the presence of impure protein, generally the enzyme isunstable above 60° C. For example, when purified enzyme obtained fromSt. morookaensis ATCC 19166 is diluted to various concentrations ofenzyme protein and heated for 10 minutes in 0.05 M glycine-NaOH buffer(pH 10) containing 5 × 10⁻³ M magnesium ion, thermal inactivation cannotbe observed even at 90° C. when the concentration of enzyme protein ishigh, but at a low concentration of enzyme protein, rapid inactivationoccurs even at 30° C. as shown in FIG. 8.

Thermal inactivation and inactivation at acidic pH can be prevented bythe addition of 20 to 300 μg/ml. of proteins such as serum albumin,casein and the like, or 0.001 to 0.01% of non-ionic surface activeagents such as Tween, Span and the like.

(e) Inhibitors:

Inhibitory actions of 40 substances which are general enzyme inhibitorssuch as nucleosides, nucleotides and their bases were examined for theireffect on the enzymes obtained from the six actinomycetes.

Among the non-substrate nucleic acids tested, guanine, guanosine, d-GDP,and d-GTP (10 mM each) produced marked inhibition, while other nucleicacids such as adenine, adenosine, 2' (3') AMP, hypoxanthine, inosine,xanthine, uracil, uridine, cytosine, cytidine, CMP and NAD were notinhibitory. Concentrations of sodium borate, 5 mM mercuric acetate, 5 mMiodoacetate and 0.1 mM N-bromosuccinimide inhibit 50 to 80% of enzymeactivity.

(f) Stimulation:

Enzyme activity can be markedly stimulated, that is, increased by theaddition of 0.05 to 0.001% of non-ionic surface active agents such asTween 20, 40 and 60, and Span 20, 40, 60 and 80 and the like,0.005-0.001% of sodium dodecyl sulfate, 0.001 to 0.01% of polyethyleneglycol 6000, 10 to 200 μg/ml. of proteins such as serum albumin andcasein and the like. Enzyme activity is further increased bypreincubating the enzyme with these activators at 30° to 0° C. for 10 to60 minutes prior to enzyme reaction.

(g) Enzyme assay:

As described above.

(h) Molecular weight:

When the purified enzymes isolated from each strain of actinomycetes areapplied to calibrated columns of Sephadex G-75 which has beenequilibrated with 0.05 M glycine-NaOH buffer, pH 9.0, the enzymeactivity is eluted as a single symmetrical peak and the apparentmolecular weight of the enzyme is calculated employing referenceproteins, namely chymotrypsin, ovoalbumin, RNase and calf serum albumin,as shown in FIG. 9.

In FIG. 9, (1) shows the molecular weight of St. morookaensis ATCC 19166enzyme, (2) is the molecular weight of St. aspergilloides ATCC 14808 andStreptoverticillium septatum ATCC 27464, (3) is the molecular weight ofSt. hachijoensis ATCC 19769, (4) is the molecular weight of Actinomycesviolascens ATCC 23968 and (5) is the molecular weight of St.adephospholyticus A 4668. ATCC 31122.

If gel-filtration is carried out in a solution of high ionic strength(0.1 M KCl), the apparent molecular weight becomes small as shown inparentheses in Table 3. Molecular weights of the enzymes are summarizedin Table 3, which also shows the results of gel-filtration and theresults obtained in polyacrylamide gel electrophoresis in 0.1% SDS usingcalf serum albumin, lysozyme and γ-globulin as references.

                  Table 3                                                         ______________________________________                                        Molecular weight                                                               Methods        Sephadex G-75                                                                            SDS-polyacrylamide                                 Strains         Gel filtration                                                                           electrophoresis                                    ______________________________________                                        Actinomyces                                                                   violascens ATCC 23968                                                                         26000      --                                                 Streptomyces                                                                  morookaensis ATCC 19166                                                                       35000(25000)                                                                             23000 - 24000                                      Streptomyces                                                                  aspergilloides ATCC 14808                                                                     32000(23000)                                                                             21000 - 23000                                      Streptoverticillium                                                           septatum ATCC 27464                                                                           32000      --                                                 Streptomyces                                                                  hachijoensis ATCC 19769                                                                       18000      --                                                 Streptomyces                                                                  adephospholiticus ATCC                                                        31122           28000 - 29000                                                                            --                                                 ______________________________________                                    

(i) Sedimentation coefficient:

Using a glycerol gradient (15-55%) centrifugation employing γ-globulin,calf serum albumin and RNase as internal standards gave sedimentationcoefficients of 2.65 for St. morookaensis ATCC 19166 enzyme, 2.45 forSt. aspergilloides ATCC 14808 enzyme, and 2.10 for St. adephospholyticusenzyme.

(j) Isoelectric point:

Isoelectric points of the purified enzymes were determined using anelectrofocusing column with an Ampholine of pH 5 to 8 (LKB InstrumentsInc.), as shown in Table 4.

                  Table 4                                                         ______________________________________                                        Isoelectric point                                                                Strains                   pH                                               ______________________________________                                        Actinomyces violascens ATCC 23968                                                                          7.5                                              Streptomyces morookaensis ATCC 19166                                                                       6.9                                              Streptomyces aspergilloides ATCC 14808                                                                     6.8                                              Streptoverticillium septatum ATCC 27464                                                                    9.2                                              Streptomyces hachijoensis ATCC 19769                                                                       8.3                                              Streptomyces adephospholyticus ATCC 31122                                                                  7.6                                              ______________________________________                                    

As described above, the physico-chemical properties of the enzyme do notcoincide with those of known enzymes, and the enzymes are confirmed as anew and characteristic type of enzyme.

A process for the production of nucleotide pyrophosphotransferase ischaracterized by cultivating microorganisms belonging to the generaStreptomyces, Streptoverticillium and Actinomyces according toconventional methods, and recovering the enzyme from culture filtrateand mycelia during the refining processes using ammonium sulfateprecipitation, solvent precipitation, extraction, chromatographyemploying ion-exchange resin, and gel filtration. Moreover, there isprovided an enzymatic method for easily producing the new nucleoside5'-phosphate (moni, di or tri) 3' (2')-diphosphates with high yields bythe transfer of pyrophosphoryl group from the 5'-position of ATP, dATPand pppApp to the 3'-position of guanosine, adenosine or inosinenucleoside phosphates (mono, di or tri).

Production of the new nucleoside 5'-phosphate (mono, di or tri) 3'(2')-diphosphates:

Various enzyme preparations such as cultured broth, culture filtrate,cell homogenate, (NH₄)₂ SO₄ ppt, fractions from ion exchange resinchromatography and gel filtration and the like containing nucleotidepyrophosphotransferase can be used for the production of the newnucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphates in thepresent invention.

The phosphate donors are limited to ATP, dATP and pppApp and phosphateacceptors are ATP, ADP, AMP, GTP, GDP, GMP, ITP, IDP, IMP and the like,corresponding to the new nucleoside 5'-phosphate 3' (2')-diphosphateformed. One or more phosphate donors and one or more phosphate acceptorscan be provided for the enzyme reaction.

An example of the composition of the reaction mixture is given asfollows:

    ______________________________________                                        250 mM glycine-NaOH buffer (pH 10.0)                                                                      6 ml.                                              50 mM ATP sodium salt      4 ml.                                              50 mM phosphate acceptor (nucleoside phosphate)                                                          4 ml.                                              50 mM MgCl.sub.2           4 ml.                                              3 units/ml. enzyme solution                                                                              6 ml.                                             Total                       24 ml.                                            ______________________________________                                    

in which the phosphate donor and acceptor can be a free acid or sodium,lithium, and potassium salts thereof, or a combination of free acid andsalts, and the ratio of the phosphate donor to the acceptor can bealtered depending upon reaction conditions and enzyme activity.

It is preferred that the enzymatic reaction is carried out under optimalconditions of pH, temperature, present or absence of divalent metalions, appropriate substrate concentration and the like whereby theenzyme has been shown to reach maximum activity. For example, theoptimal range of pH is from 6 to 11, the optimal temperature range isfrom 25° to 40° C. and the divalent metal ions employed are Fe⁺⁺, Mn⁺⁺,Mg⁺⁺, Co⁺⁺, Zn⁺⁺ and the like. Generally the enzyme reaction is carriedout at 37° C. for 2 to 4 hours.

After termination of the enzyme reaction, the reaction mixture isneutralized with 0.1 N hydrochloric acid, and the new purine nucleoside5'-phosphate (mono, di or tri) 3' (2')-diphosphates are isolated andpurified by a combination of conventional refining methods such asadsorption on active carbon, chromatography by anion or cationion-exchange resins, solvent precipitation and the like. For example,the reaction mixture as described above is incubated at 37° C. for 4hours, neutralized with 0.1 N HCl and applied to an ion exchange resincolumn (Dowex-1, Cl type, DEAE-Sephadex A25). Adsorbed reaction productswere eluted with a proper buffer (diluted HCl or diluted HCl + NaCl forDowex 1, Cl type to prepare sodium salt, and Tris-HCl+LiCl forDEAE-Sephadex to prepare lithium salt), and the eluate was adsorbed onactive carbon and eluted again with ammonia alcohol (50% ethanol-1.4%ammonia water, ammonia-acetone or 2.8% ammonia water).

After the excess ammonia in the eluate was removed by concentration invacuo, the sodium or lithium salts of the new nucleoside 5'-phosphate 3'(2')-phosphates were obtained as a white microcyrstalline powder byallowing the solution to stand in the cold, or by the addition ofsolvents such as alcohols, acetone and the like. To prepare the freeacid, the sodium or lithium salt of the nucleoside 5'-phosphate 3'(2')-phosphate is dissolved in acidic water and adsorbed on activecarbon. Then the free acid is eluted with ammonia alcohol (50%ethanol-1.4% ammonia water) and after the excess ammonia is removed byevaporation in vacuo, the free acid is precipitated with 10 volumes ofacetone.

The structure of the new nucleoside phosphate:

The structures of the new nucleoside 5'-phosphate 3' (2')-phosphate inthe present invention are elucidated by UV adsorption spectrum, totalphosphate determination, NMR analysis, and enzymatic degradation asfollows:

For example, when the reaction mixture in which ATP was incubated withthe enzymes in the presence of Mg⁺⁺ is subjected to and adsorbed on anion-exchange DEAE-Sephadex A-25 column (Cl⁻), and eluted with a bufferwhere the lithium chloride concentration is increased from 0.1 to 0.3 Mlineally, two peaks at OD_(260nm) are eluted by 0.1 M and 0.2 M lithiumchloride. These fractions are pooled and concentrated in vacuo, and thentwo kinds of nucleotides are precipitated by the addition of 10 volumesof 95% ethanol. From the results of periodate oxidation, total phosphatedetermination, ultra violet spectrum, ¹³ C-NMR analysis and enzymaticdegradation by snake venom dephosphodiesterase and 3'-nucleotidase, thefirst peak was determined to be AMP lithium salt and the second peak wasconfirmed to be a new adenosine 5'-triphosphate 3' (2')-diphosphatelithium salt, which has a pyrophosphate structure attached to the 3'(2')-position of ATI as follows (I): ##STR1##

To characterize and identify the new nucleoside 5'-phosphate (mono, dior tri) 3' (2')-diphosphates produced by the methods of the presentinvention, the results of lithium content, spectral properties of UV andNMR, and molar ratio of Pi and nucleotide calculated by the values oftotal phosphate and ribose contents are summarized in Table 5. Further,UV adsorption and NMR spectra of typical nucleoside 5'-phosphate (mono,di or tri) 3' (2')-diphosphates according to the present invention areshown in FIGS. 1 to 6 to clarify the characteristics of these compounds.

                                      Table 5                                     __________________________________________________________________________            Nucleoside 5'-phosphate 3'-diphosphate lithium salt                   Properties                                                                            pppApp                                                                              ppApp                                                                              pppGpp                                                                             ppGpp                                                                              pGpp pppIpp                                      __________________________________________________________________________    Ultraviolet                                                                   adsorption                                                                    (Remarks 1)                                                                   λmax (1)                                                                       258   258  258  258  257.5                                                                              250                                         (2)     260.5 260  254.5                                                                              254.5                                                                              253.5                                                                              249.5                                       (3)     260.5 260  258  258  258  254.5                                       λmin (1)                                                                       230.5 230.5                                                                              229  229  229  221.5                                       (2)     228   228  224  224  223.5                                                                              223                                         (3)     228   228  226  225.5                                                                              225.5                                                                              225                                         ε260nm 10.sup.-3                                                      (1)     14.3  14.2 11.8 11.8 11.8 7.4                                         (2)     15.1  15.0 12.0 11.9 12.1 7.4                                         (3)     15.1  15.0 11.8 11.8 11.8 12.2                                        Phosphate                                                                     content                                                                       (molar ratio)                                                                         1:4.92                                                                              1:4.00                                                                             1:4.95                                                                             1:3.82                                                                             1:3.02                                                                             1:4.69                                      Lithium                                                                       content                                                                       (molar ratio)                                                                         1:7.1 1:6.0                                                                              1:6.8                                                                              1:6.2                                                                              1:5.2                                                                              1:7.2                                       Binding pos-                                                                  ition of -phosphate                                                           (Remarks 4)                                                                           5'-, 3'-                                                                            5'-  5'-  5'-  5'-  5'-                                                       3'-  3'-  3'-  3'-  3'-                                         __________________________________________________________________________

Remarks 1: UV absorption was measured in (1) 0.01 N HCl, (2) distilledwater and (3) 0.01 N NaOH.

Remarks 2: Total phosphate determination was carried out by Nakamura'smethod after 0.5 to 3 ml. of sample solution was degraded with 0.5 ml.of 60% perchloric acid, heated for one hour and made up to 10 ml. totalvolume with distilled water. Inorganic phosphate in a degraded samplewas determined from optical density at 700 nm using a standard curve ofKH₂ PO₄, after adding 0.5 ml. of 1.5% H₂ SO₄, 0.5 ml. of 3.3% ammoniummolybdate and 0.5 ml. of amidol reagent to 3.5 ml. of degrading solutionand permitting the sample to stand for 20 minutes at 25° C.

Remarks 3: Lithium content was determined by flame photometry usingJarrell Ash Flame Emission Spectrophotometer Model AA-780; C₂ H₄ (0.4kg/cm²) 1.75 l/min; air (1.5 kg/cm²), 9.0 l/min; sensitivity, 3.90; wavelength, 2708A.

Remarks 4: Assignment of structure and the binding position ofpyrophosphate to the nucleoside by NMR spectroscopy.

The sample nucleoside 5'-phosphate 3'-(2')-phosphates were dissolved inD₂ O. Proton noise-decoupling ¹³ C-NMR spectra were obtained at 25.2 MHzon a Varian XL-100-15 spectrophotometer operating in the Fouriertransform mode at room temperature. All chemical shifts were calculatedrelative to an internal reference, dioxane.

Proton-NMR spectra were recorded on the same instrument operating in theFourier transform mode at 100 MHz. Chemical shifts were recorded from aninternal 2,2-dimenthyl-2-silapental-5-sulfonate.

The present invention has further provided a new nucleoside 5'-phosphate(mono, di or tri) 3' (2')-monophosphates, derived from nucleoside5'-phosphate (mono, di or tri) 3' (2')-diphosphates and the process forthe production of these additional nucleosides.

When nucleoside 5'-phosphate (mono, di or tri) 3' (2')-diphosphates aretreated in 0.1 N HCl or 0.1 N NaOH in the presence of a barium salt for1 to 2 hours at 37° C. and purified by combination of adsorption onactive carbon, solvent precipitation and ion-exchange resinchromatography, the new nucleoside 5'-phosphate (mono, di or tri) 3'(2')-monophosphates (II) are easily obtained as a white microcrystallinepowder, as the result of the release of one phosphate frompyrophosphoryl group at 3'-position of nucleoside 5'-phosphate (mono, dior tri) 3' (2')-diphosphates. ##STR2##

Biological activity of new nucleoside 5'-phosphate (mono, di or tri)3'-diphosphates and nucleotide pyrophosphotransferase:

The characteristic of the said nucleotide pyrophosphotransferase is thatit inhibits the growth of Hela S₃, L1210 and L5178Y cells in culture ata relatively low concentration, 5 to 10 units/ml. When these cells areincubated with various concentrations of the purified enzyme obtainedfrom St. adephospholyticus A 4668 in a proper medium, RPMI 1640-20% calfserum for L5178Y and L1210 cells and MEM - 10% calf serum for Helacells, the growth of cells is markedly inhibited, being accompanied bythe inhibition of protein synthesis, as shown in Table 6. Furthermore,antitumor action can be most significantly demonstrated in experimentaltumors in mice. For example, when BDF₁ mice weighing 18 to 22 grams wereintraperitoneally inoculated with 2 × 10⁶ cells of L1210 cells and thenucleotide and the enzyme was administered intraperitoneally once dailyfor 10 days consecutively beginning 24 hours after the inoculation, theenzyme and pppApp distinctly suppressed the accumulation of abdominaldropsy without causing toxicity to the mice and prolonged the lifespanas shown in Table 7, but ATP and heat-inactive enzyme are not effectiveon the tumor-bearing mice. The nucleotide or enzyme is combined with aconventional pharmaceutically acceptable nontoxic carrier forintraperitoneal administration.

                  Table 6                                                         ______________________________________                                        Effect of purified nucleotide pyrophosphotransferase                          from St. adephospholyticus A 4668 on the cell growth                          and protein synthesis                                                                   Inhibition (% of control)                                           Enzyme                         Protein                                        concentration                                                                          cells  Cell growth        Synthesis                                  (units/ml.)     Hela    L5178Y  L1210  L1210                                  ______________________________________                                        0           0       0         0      0                                        1           0       9         12     24                                       5           1       30        29     35                                       10          25      92        63     59                                       ______________________________________                                    

                  Table 7                                                         ______________________________________                                        Effects of nucleotide and purified nucleotide                                 pyrophosphotransferase on the life span of                                    mice                                                                                     Dose        MST      T/C   30 days                                 Substances (mg/kg.day) (day)    (%)   survivors                               ______________________________________                                        pppApp 7Li 20          16.8     189   2/5                                                10          12.5     140   1/5                                                5           14.0     157   1/5                                     ATP 2Na    20          8.6      97    0/5                                                10          9.2      103   0/5                                                5           8.7      98    0/5                                     Native enzyme                                                                            2           13.4     151   1/5                                     (specific activity                                                            1293)      1           13.8     155   0/5                                                0.5         12.2     137   0/5                                     Heat-inactive                                                                            2           9.8      110   0/5                                     enzyme                                                                        (no activity)                                                                            1           10.6     119   0/5                                                0.5         9.0      101   0/5                                     Control    --          8.9      100    0/10                                   ______________________________________                                         Animal: Male BOF.sub.1                                                        Tumor: L1210, Sloan-Kettering line, 10.sup.5 cells/head, i.p.                 MST: Mean survival time                                                  

The following examples, in which proportions are by weight unlessotherwise indicated, illustrate methods of carrying out the presentinvention, but it is to be understood that they are given for purposesof illustration and not limitation.

EXAMPLE 1

An aqueous medium having the following composition and pH was prepared:

    ______________________________________                                                            Percent                                                   ______________________________________                                        glycerol              2.0                                                     polypeptone           4.0                                                     KH.sub.2 PO.sub.4     0.1                                                     K.sub.2 HPO.sub.4     0.1                                                     MgSO.sub.4 . 7H.sub.2 O                                                                              0.05                                                   Mn.sup.++ (as MnSO.sub.4)                                                                           2 ppm                                                   Fe.sup.++ (as FeSO.sub.4)                                                                           2 ppm                                                   pH                    7.0                                                     ______________________________________                                    

100 ml. of this medium was sterilized at 120° C. in a 500 ml.Sakaguchi-shaking flask which was inoculated with a culture ofStreptomyces asperigilloides ATCC 14808, and incubated at 28° C. for 2days on a shaker. 10 liters of this medium in a 20-liter stainless steeljar fermentor were aseptically inoculated with 1¢ by volume of a growingculture described above. The fermentation was carried out at 28° C. for40 hours with agitation and aeration. After the mycelia were removedfrom the cultured broth by filtration, ammonium sulfate was added to thefiltrate (1), giving a 60% saturation, the solution was mixed andallowed to stand overnight in the cold. The resulting precipitate (2)was harvested by centrifugation, dissolved in water and dialyzed for 24hours against running water. Any precipitate that appeared duringdialysis was removed by centrifugation. The dialyzed preparation wasapplied to a DEAE-cellulose column which had been equilibrated with 0.01M acetate buffer (pH 5.5), and the enzyme passed through the columnwithout retention. The active fraction (3) was applied to a CM-SephadexC-25 column which had been equilibrated with 0.01 M acetate buffer (pH5.5) containing 0.001 M MgCl₂. After the column was washed with the samebuffer, the enzyme was eluted lineally with the same buffer supplementedwith 0.1 to 0.5 M NaCl. The active fraction eluted with 0.35 to 0.45 MNaCl (4) was concentrated by the addition of ammonium sulfate (60%saturation). The precipitated enzyme was dissolved in a small amount of0.01M Tris-HCl buffer (pH 8.7) containing 0.01 M MgCl₂, dialyzedsufficiently against the same buffer, and applied to a DEAE-SephadexA-50 column which had been equilibrated with 0.01 M Tris-HCl (pH 8.7)containing 0.01 M NaCl. After the column was washed with the samebuffer, the enzyme was eluted in stepwise fashion with 0.02, 0.05 and0.15 M NaCl.

The active fraction eluted with 0.05 M NaCl (5) was dialyzed against0.01 M Tris-HCl buffer (pH 8.7) applied to a hydroxylapatite columnwhich had been equilibrated with the same buffer, and eluted with linearpH gradient (pH 8 to 9) of the buffer.

The active enzyme fraction eluted at pH 9 to 8.7 (6) was then applied toa Sephadex G-75 column which had been equilibrated with 0.05 Mglycine-NaOH buffer (pH 9.0) containing 0.01 M MgCl₂.

Gel filtration was carried out with the same buffer and a homogeneousenzyme solution (7) was obtained. The results of enzyme purification arepresented in Table 8.

                  Table 8                                                         ______________________________________                                                        Total    Total                                                Active Volume   activity protein  Specific                                                                             Yield                                fraction                                                                             (ml.)    (units)  (OD.sub.280nm)                                                                         activity                                                                             (%)                                  ______________________________________                                        (1)    10000    245000   252000   0.97   100                                  (2)    600      80000    32000    2.5    32.5                                 (3)    1500     75000    7000     10.7   30.6                                 (4)    800      65000    1850     35.2   26.5                                 (5)    600      60000      720    84.5   24.5                                 (6)    110      33000      178    186    13.5                                 (7)     45      19500     25      780    8.0                                  ______________________________________                                    

EXAMPLE 2

Streptomyces morookaensis ATCC 19166 and Actinomyces violascens ATCC23968 were cultivated separately under the conditions of Example 1. Ineach case, 1000 ml. of culture filtrate was treated according to thepurification procedures of Example 1, and the purified enzymes wereobtain as shown in Table 9.

                  Table 9                                                         ______________________________________                                                       Actinomyces  Streptomyces                                                     violascens   morookaensis                                      Strain No.     ATCC 23968   ATCC 19166                                        ______________________________________                                        Filtrate                                                                             Total activity                                                                (units)     31000        42500                                                Specific                                                                      activity    0.81         0.58                                          Purified                                                                      enzyme:                                                                              Total activity                                                                (units)     2050         3650                                                 Yield (%)   6.6          8.6                                                  Specific                                                                      activity    1100         960                                           ______________________________________                                    

EXAMPLE 3

An enzyme reaction mixture having the following composition wasprepared:

    ______________________________________                                        0.25M Tris-HCl buffer (pH 9.0)                                                                         160 ml.                                              0.05M MgCl.sub.2         20 ml.                                               ATP . 2N                 2.5 g                                                Distilled water          20 ml.                                               Total                    200 ml.                                              ______________________________________                                    

A small collodion bag containing 10 ml. of purified enzyme solution(specific activity: 274, total units: 465) which was obtained fromStreptomyces morookaensis ATC 19166 was placed in the reaction mixturedescribed above, and incubated at 30° C. for 24 hours with stirring.

100 ml. of the reaction mixture was diluted to twice the volume adjustedto pH 7.8 with HCl, and adsorbed on a DEAE-Sephadex A-25 (3 × 55 cm)column which had been equilibrated with 0.01 M Tris-HCl buffer (pH 7.8).The column was washed with the same buffer and then pppApp was elutedwith a salt linear gradient of 0.15 to 0.35 M LiCl in the same buffer.

No. 95 to 124 fractions (20 ml. each) eluted with the saltconcentrations of 0.22 to 0.25 M were pooled, concentrated at 37° to 42°C. in vacuo, and then precipitated by the addition of 5 volumes ofacetone:ethanol mixture (1:1 by volume). A pppApp Li salt was washedwith 95% by volume ethanol five times, and 1.55 g of this salt wasobtained after drying in vacuo.

EXAMPLE 4

The reaction mixture consisted of 250 mM glycine-NaOH buffer (pH 10.0),6.0 ml.; 100 mM ATP, 2.0 ml.; 50 mM MgCl₂, 4.0 ml.; 100 mM pyrophosphateacceptor indicated in Table 10, 2.0 ml.; distilled water, 4.0 ml.; andenzyme solution obtained from the various purification steps of theculture broth of Streptomyces aspergilloides ATCC 14808, 6.0 ml. Thereaction mixture was incubated at 37° C. for two hours, adsorbed on aDowex 1 × 4, Cl type, column (1.7 × 25 cm) which had been equilibratedwith 0.01 M NaCl in 0.01 N HCl, and then eluted with a linear gradientfrom 0.01 to 0.5 M NaCl in 0.01 N HCl. Fractions eluted at theconcentrations of 0.2 to 0.25 M NaCl were pooled, adjusted to pH 7 with0.1 N NaOH, and readsorbed on a DEAE-Sephadex A-25 (2.2 × 25 cm) columnwhich had been equilibrated with 0.01 M Tris-HCl buffer (pH 7.5). Byeluting with 0.1 to 0.3 M LiCl, fractions having an optional density of260 nm were pooled, concentrated 37° C. in vacuo, and white precipitatewas obtained by the addition of 10 volumes of 99% by volume ethanol. Theprecipitate was dissolved in a small amount of water and 5 volumes of90% by volume ethanol were added thereto to reprecipitate theprecipitate. After drying the reprecipitated precipitate in vacuo,nucleotide lithium salt was obtained as indicated in Table 10.

                                      Table 10                                    __________________________________________________________________________                                      Yield                                                                         (mg as                                                Spcific                                                                            Photphate                                                                           Phosphate                                                                            Main  lithium                                     Enzyme sources                                                                          activity                                                                           donor acceptor                                                                             product                                                                             salt)                                       __________________________________________________________________________    Purified enzyme                                                                         650  ATP   ATP    pppApp                                                                              115                                         "         "    ATP   ADP    ppApp 87                                          "         "    ATP   GTP    pppGpp                                                                              135                                         "         "    ATP   GDP    ppGpp 120                                         "         "    ATP   ITP    pppIpp                                                                              62                                          "         "    pppApp                                                                              AMP    pApp  85                                          Culture filtrate                                                                        0.21 ATP   ATP    pppApp                                                                              75                                          DEAE-cellulose                                                                chromatography                                                                active fraction                                                                         1.95 ATP   ATP    pppApp                                                                              125                                         CM-Sephadex                                                                   chromatography                                                                active fraction                                                                         53.5 ATP   ATP    pppApp                                                                              108                                         DEAE-Sephadex                                                                 chromatography                                                                active fraction                                                                         182  ATP   ATP    pppApp                                                                              132                                         "         "    dATP  GDP    ppGpp 86                                          "         "    ATP   GTP    pppGpp                                                                              125                                         "         "    ATP   GMP    pGpp  74                                          "         "    pppApp                                                                              GMP    pGpp  26                                                                      pApp  38                                          __________________________________________________________________________

EXAMPLE 5

Purified enzyme was obtained from the culture filtrate of Streptomycesadephospholyticus A 4668, which was cultivated under the fermentationconditions of Example 1, according to the methods described inExample 1. 20 times volumetric diluent of this purified enzyme (specificactivity: 1500, 60 units/ml.), 6.0 ml., was incubated with 5 ml. of 1 MTris-HCl buffer (pH 10.0), 500 mg of ATP.2Na, 100 mg of ADP.2Na, 5.0 ml.of 0.05 M MgCl₂ and 10 ml. of distilled water, for two hours at 37° C.During the enzyme reaction, pH was controlled at 8.5 by addition of 1 NNaOH. The reaction mixture was applied to a DEAE-Sephadex A-25 column(2.2 × 25 cm) which had been equilibrated with 0.01 M Tris-HCl buffer(pH 7.2), eluted with a linear gradient from 0.1 to 0.3 M LiCl in thesame buffer, and fractionated every 15 ml. Resulting pppApp and ppAppfractions were concentrated to one twentieth by volume in vacuo andadded thereto were 5 volumes of cold acetone:ethanol mixture (9:1 byvolume). About 250 mg of the precipitate obtained by filtration weredissolved in 100 ml. of water, adsorbed on a DEAE-Sephadex A-25 columm(1.8 × 35cm), and then eluted with 0.15 to 0.35 M LiCl solution. pppAppand ppApp fractions were concentrated to one twentieth by volume at 37°C., in vacuo and precipitated by addition of 5 volumes of coldacetone:ethanol mixture (9:1 by volume). After several washings with thesame mixture and drying in vacuo, 70.5 mg of ppApp.6Li and 142 mg ofpppApp.7Li were obtained as white microcrystalline powder.

EXAMPLE 6

Reaction mixture consisting of 250 mM glycine-NaOH buffer (pH 10.0), 6.0ml.; 50 mM ATP, 4.0 ml.; 50 mM GMP, 4.0 ml.; 50 mM MgCl₂, 4.0 ml. andthe enzyme solution (specific activity: 150, 3 units/ml.) obtained fromStreptoverticillium septatum ATCC 27464, 6.0 ml. was incubated at 37° C.for 4 hours, applied to a Dowex 1 × 4, Cl type, column (1.7 × 20 cm),and then eluted with a linear gradient from 0.01 to 0.5 M NaCl in 0.01 NHCl. Fractions eluted between 0.21 and 0.24 M NaCl were pooled,neutralized with 0.1 N NaOH, and adsorbed on a DEAE-Sephadex A-25 column(2.2 × 25 cm) which had been equilibrated with 0.05 M Tris-HCl buffer(pH 7.5). 200 ml. of pGpp fraction eluted with 0.1 to 0.3 M LiCl wereconcentrated to 10 ml. in vacuo, and added thereto were 10 volumes of99% by volume ethanol. The resultant precipitate of pGpp lithium saltwas dissolved in a small amount of water, and reprecipitated with 90% byvolume ethanol. After drying in vacuo, 56.2 mg of pGpp.5Li were obtainedas white microcrystalline powder.

EXAMPLE 7

200μ mole of ATP or dATP and 200μ mole of various nucleotides wereincubated using the purified enzyme, the reaction mixture compositionand the reaction conditions described in Example 4. Each nucleotide wasrecovered from the reaction mixture according to the methods forisolation and purification of Example 5 and 6. Main reaction productsand their yield are shown in Table 11.

                  Table 11                                                        ______________________________________                                                    Phosphate             Yield (mg                                   Phosphate donor                                                                           acceptor  Main product                                                                              as Li salt)                                 ______________________________________                                        ATP         ATP       pppApp      132                                         "           ADP       ppApp       105                                         "           GTP       pppGpp      140                                         "           GDP       ppGpp       130                                         "           GMP       pGpp        52.5                                        "           ITP       pppIpp      74.2                                        "           IDP       ppIpp       25.6                                        dATP        GTP       pppGpp      92.5                                        "           ADP       ppApp       69.3                                        pppApp      GMP       pGpp        22.5                                                              pApp        18.3                                        ______________________________________                                    

EXAMPLE 8

20 mg of each nucleoside 5'-phosphate (mono, di or tri) 3'-diphosphateslithium salts obtained in Example 7 was weighed, dissolved in 20 ml. of0.25 N HCl, and the solution was allowed to stand at 37° C. for onehour. After being neutralized with 0.5 N KOH solution, the solution wasrefined by the methods of ion-exchange resin column chromatography,adsorption onto active carbon, and solvent precipitation as described inExamples 1 and 2, which resulted in white microcrystalline powders oflithium salts as follows:

                  Table 12                                                        ______________________________________                                        Starting                Yield                                                 materials  Products     (mg as Li salt)                                       ______________________________________                                        pppApp     ppAp         11.2                                                  ppApp      ppAp         13.8                                                  pppGpp     pppGp        9.8                                                   ppGpp      ppGp         7.5                                                   pGpp       pGp          6.3                                                   pppIpp     pppIp        4.6                                                   ______________________________________                                    

What is claimed is:
 1. A purine nucleoside 5'-(mono, di or tri)phosphate 3'(2')-diphosphate and the corresponding purine nucleoside5'-(di or tri) phosphate 3'(2')-monophosphate and the salts thereofselected from the group consisting ofadenosine-5'-monophosphate3'(2')-diphosphate(pApp), adenosine-5'-diphosphate3'(2')-diphosphate(ppApp), adenosine-5'-triphosphate3'(2')-diphosphate(pppApp), guanosine-5'-monophosphate3'(2')-diphosphate(pGpp), inosine-5'-monophosphate3'(2')-diphosphate(pIpp), inosine-5'-diphosphate3'(2')-diphosphate(ppIpp), inosine-5'-triphosphate3'(2')-diphosphate(pppIpp), β,γ-methylene adenosine 5'-triphosphate3'(2')-diphosphate(p(CH₂)ppApp), guanosine-5'-triphosphate3'(2')-monophosphate(pppGp), inosine-5'-diphosphate3'(2')-monophosphate(ppIp), inosine-5'-triphosphate3'(2')-monophosphate(pppIp), β,γ-methylene adenosine-5'-triphosphate3'(2')-monophosphate(p(CH₂)ppAp) and β,γ-methyleneguanosine-5'-triphosphate 3'(2')-monophosphate(p(CH₂)ppGp) and lithium,potassium and sodium salts thereof.
 2. A pharmaceutical composition forintraperitoneal administration to animals comprising a substanceaccording to claim 1 in a concentration sufficient to suppress anaccumulation of abdominal dropsy without causing toxicity to therecipient in vivo in combination with a pharmaceutically acceptablenontoxic carrier.
 3. A method for chemotherapeutically treating ananimal bearing ascites tumor including an accumulation of abdominaldropsy, which comprises administering a compound according to claim 1 tosaid animal in a dosage sufficient to lower said accumulation withoutcausing toxicity to said animal.
 4. A pharmaceutical composition ofclaim 2 wherein said animals are rodents.
 5. A method of claim 3 whereinsaid animal is a rodent.